Identification of Nucleocapsid Binding Sites within Coronavirus-Defective Genomes

Abstract

The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N-viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3' end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949-4524), as well as a region at the 3' end of the MHV N ORF (nts 4837-5197) within MIDI-C. Binding to the full-length MIDI-C transcript (approximately 5500 nts) and to an approximately 1-kb transcript from the gene 1a region (nts 935-1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.

Recombinant Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Forms a Dimer through Its C-terminal Domain

“…The oligomerization states of coronavirus N proteins are reported to be dimeric (36), trimeric (16,30), and higher (37). In this report, three independent methods, analytical ultracentrifugation, SEC-LS, and chemical cross-linking, were used to study the self-association of the SARS-CoV N protein.…”

“…Nonspecific RNA and DNA binding by native CoV N protein was reported previously (16,18,29,30). Thus, oligonucleotide binding was used as a test for proper folding of the recombinant protein.…”

RNA Binding Domain of Jamestown Canyon Virus S Segment RNAs

“…The crystallized nucleocapsid of another negativesense RNA virus, influenza virus, which also has the hallmark ability to form a panhandle structure from complementary ends, illustrates that binding with its RNA occurs in a singlestranded manner rather than being double stranded (32). Other encapsidation signals from a variety of viruses have shown the importance of stem-loops for binding (1,(3)(4)(5) but not the importance of double-stranded RNA.…”

Mouse Hepatitis Coronavirus Nucleocapsid Phosphorylation

“…A rabbit polyclonal N antibody was used to identify the N protein (Cologna et al, 2000). Knowing where phosphorylated sites are located on the protein will help direct future molecular studies to address howthe modification contributes to or modulates functions of the multifunctional N protein.…”